CD123+ dendritic cells in blood and lymphoid tissues

ABSTRACT

Dendritic cells (DCs) are the primary antigen presenting cells during the initiation of T cell-dependent immune responses. The cells originate from the bone marrow and have been suggested to represent a distinct cell lineage. However, distinct DC precursors have not been identified in bone marrow, and mature monocytes can also give rise to DCs. The instant invention presents a distinct DC precursor among bone marrow CD34 +  cells. The cells express high levels of the interleukin-3 receptor α chain and CD4 and can be uniquely identified also in blood and lymphoid tissues by this phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application No. 08/939,545filed Sep. 29, 1997, now U.S. Pat. No. 6,008,004, which claimed thebenefit of U.S. Provisional Patent Application No. 60/027,898, filedOct. 4, 1996, now abandoned.

BACKGROUND OF INVENTION

Dendritic cells (DCs) play a key role in the immune system as initiatorsof primary T cell responses. DCs are difficult cells to isolate becausethey are sparsely distributed in the tissues, and because there is alack of positive markers identifying the cells. There is, thus, littleinformation concerning DCs in various organs, and, while variousresearchers have postulated on the origin of these cells, it is notdefinitively known whether these cells constitute a separate lineage ofcells. Furthermore, while there are many theories regarding this, it isnot known if there is a dendritic committed progenitor cell in the bonemarrow. The importance of solving these questions has become evidentfrom a clinical perspective, as their function has made them intocritical targets for vaccine development against tumor cells, and,potentially, in the development of vaccines against HIV infection.

Furthermore, since some DCs have been observed to serve as reservoirsfor the HIV virus, identification of subtypes of cells havingdifferences in infectability potential and/or the ability to provideinfection resistance, is becoming increasingly desirable. In addition,there is considerable interest in identifying subsets of DCs believed tobe involved in the induction of donor-specific tolerance aftertransplantation. Thus, there exists a need for methodologies tocharacterize and isolate dendritic cells.

Such methodologies would also be useful in the isolation of DCs byutilization of the phenotype by use of cytometry. Flow cytometry isparticularly useful in this regard, as the cells bearing a particularphenotype can be recognized and sorted by use of labeled antibodies.Thus, DCs of a known phenotype could be sorted and isolated.

SUMMARY OF INVENTION

A CD34⁺ (as used herein, any cell designated as positive (⁺) for aparticular marker will be a cell which expresses that marker asevidenced by the binding of an antibody directed against the recitedmarker; a cell designated as negative (−) does not express the marker asmeasured by the same methodology) mammalian, preferably human, bonemarrow precursor of a distinct type of DCs that can be identified inperipheral blood, thymus, spleen, tonsil and lymph nodes solely by thepositive expression of CD123 and CD4 has been characterized. This CD34⁺population of bone marrow cells contains primitive progenitor cellscapable of reconstituting the entire hematopoietic system and moredifferentiated progenitors committed to a single lineage. Subsets ofprogenitor cells can be identified by specific cell surface markers. Itis known that the receptor for macrophage-colony-stimulating factor(M-CSFR/CD115) is expressed on granulomonocytic progenitors, but not onprimitive, erythroid, or CD19⁺ B-lymphoid progenitors. A population ofCD34^(lo) (as used herein, the magnitude of expression of a particularantigen can be designated as lo or hi; such cells express the antigen,as measured by the binding of an antibody directed against the recitedantigen, to a lesser (for lo) or greater (for hi) magnitude than atleast 50%, preferably 75%, more preferably 90% of the cells which arepositive for the recited antigen) cells (3±0.8% of the CD34⁺ cells) wasidentified that stained brightly with antibodies to the interleukin 3receptor alpha chain (IL3R alpha/CD123) and weakly with antibodies tothe M-CSFR (see FIGS. 1A and 1B). Similar cells could be generated byshort term culture of M-CSFR⁺ cells, suggesting that the cells belong tothe granulomonocytic lineage (see FIG. 1C). Unlike most granulomonocyticprogenitors in fetal bone marrow, the CD123^(hi) cells had low levels ofCD64 and CD13, but expressed high levels of CD36, a molecule found onmonocytic cells. Further characterization showed that the cells hadseveral phenotypic characteristics of DC precursors isolated fromperipheral blood (CD3⁻, CD4⁺, CD11c⁻, CD13^(lo), CD14⁻, CD16⁻, CD19⁻,CD33⁺, CD45RA⁺, CD45RO⁻, CD56⁻, CD64⁻, HLA-DR⁺). Morphologically thecells had a smooth plasma membrane, abundant agranular cytoplasm andreniform or slightly lobulated nucleus (see FIG. 1D).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D present dot plots of CD34 (FIG. 1A) and IL-3R alpha (FIG. 1Band FIG. 1C) versus M-CSFR expression of isolated CD34^(lo) CD123^(hi)cells; FIG. 1D presents a micrograph of these cells. Cells were stainedwith fluorescein isothyocyanate (FITC), phycoerythrin (PE) orallophycocyanin (APC) as indicated.

FIG. 2A presents a dot plot of CD1a versus CD14 expression of CD34^(lo)CD123^(hi) cells cultured for 7 days; FIGS. 2B and 2C presentmicrographs of these cells cultured only in the presence of GM-CSF; FIG.2D demonstrates the reactivity of these cells as activators of CD4⁺allogeneic T-cells.

FIGS. 3A-3B present dot plots of side scatter versus CD123 expression(FIG. 3A) and HLA-DR expression (FIG. 3B). Cells were stained withperidinin chlorophyll protein (PerCP) as indicated.

FIGS. 4A-4B present dot plots of CD123 expression versus HLA-DRexpression for adult tonsil (FIG. 4A) and fetal lymph node (FIG. 4B)derived cells.

DETAILED DESCRIPTION OF INVENTION

To examine the developmental potential of CD34⁺ CD123^(hi) cells, thecells were sorted using a flow cytometer and fluorescently labeledantibodies using techniques well-known in the art, and cultured with awide range of cytokines capable of supporting multiple hematopoieticlineages [granulocyte-macrophage colony-stimulating factor (GM-CSF),G-CSF, M-CSF, interleukin-3 (IL-3), IL-6, stem cell factor (SCF) anderythropoietin (Epo)]. Cells from freshly isolated bone marrow and thosegenerated in vitro from CD34^(hi) CD115⁺ cells gave similar results, butthe viability was better for the latter. Sorted cells rapidly formedlarge aggregates after a few hours in liquid culture, a growth patternwhich is characteristic of DCs. After 7 days the cells were positive forthe DC marker CD1a, whereas markers for monocytes (CD14) andgranulocytes (CD15) were absent or expressed at low density (see FIG.2A). Wright-Giemsa-stained cytospin preparations showed that the cellshad abundant, agranular, cytoplasm with dendritic protrusions, and roundnuclei. The CD34⁺ CD123^(hi) cells expanded on average only 3-foldduring this culture period, and no further cell expansion occurredbetween 7 and 20 days of culture under these conditions.

The expression of CD1a could indicate that the cells were DCs of theLangerhans type. However, electron microscopy of freshly isolated andresorted CD34⁺ CD123^(hi) cells cultured 7 days in GM-CSF and TNF alphagenerated cells lacking Birbeck granules, but with a dendriticmorphology of lobulated nucleus and a villous surface. The CD34⁺CD123^(hi) cells assumed dendritic morphology also when cultured withGM-CSF as the only added cytokine (see FIGS. 2B and 2C), and expressedhigh levels of HLA-DR and the costimulatory molecule CD80. These cellswere potent activators of allogeneic CD4⁺ T cells compared tomacrophages (see FIG. 2D).

A distinct population of CD123^(hi) cells was also observed amongperipheral blood mononuclear cells (see FIG. 3A). Among these, 60% werenegative for HLA-DR and CD4 (within region in FIG. 3B), positive foranti-human IgE and CD11b and had morphological characteristics ofbasophilic granulocytes. However, 40% expressed high levels of moleculestypically expressed by DCs, such as CD4, HLA-DR (MHC class II) (see FIG.3B), the costimulatory molecules CD40 and CD86 and the adhesionmolecules CD54 (ICAM-1) and CD58, but with low or negative expression ofthe costimulatory molecule CD80. The cells furthermore expressed theDC-associated marker CMRF-44, and had almost identical immunophenotypeto the CD34⁺ CD123^(hi) cells (CD3⁻, CD11b⁻, CD11c⁻, CD13^(lo) , CD14⁻,CD15⁻, CD16⁻, CD19⁻, CD33^(lo), CD45RA⁺, CD45RO, CD56⁻, CD64⁻). TheCD123^(hi) CD4⁺ cells had agranular cytoplasm and a reniform orlobulated nucleus, similar to the CD34⁺ CD123^(hi) cells. In culture,the cells formed large aggregates similar to those observed duringculture of bone marrow cells. When allowed to differentiate for 36 h inculture, the CD123^(hi) HLA-DR⁺ cells induced extensive proliferation ofboth allogeneic and autologous T cells compared to monocytes. The cellstherefore have similar morphology, phenotype and functionalcharacteristics as a population of peripheral blood DCs described inseveral earlier publications. Our results provide evidence to supportthe hypothesis from those studies that these DCs represent immaturecells in transit from bone marrow to tissues.

In addition to the population described above, peripheral blood containsseveral other cell types that can give rise to DCs in vitro, includingCD34⁺ cells, mature monocytes and a population of HLA-DR^(hi) CD11^(hi)cells that express low levels of a number of lineage markers for B cell,T cells, NK cells, and monocytes. The relationship between eachpopulation and DCs in various tissues has been difficult to establish,as several DCs change phenotypic characteristics during migration. Celltracking experiments in rats have, however, shown that some immature DCsrapidly migrate via blood and lymphatics to T cell-dependent areas ofsecondary lymphoid tissue. While not wishing to be bound by theory, itis hypothesized that CD123^(hi) CD4⁺ cells may have a similar migratorypathway and enter tonsils and spleen with little change in phenotype.

Tonsillar DCs were identified as cells with high expression of HLA-DRand low levels of lineage markers for B cells, T cells, NK cells andmonocytes. Cells with these characteristics constituted 0.2-0.4% of thetotal mononuclear cell population, and interestingly the majority wereCD123^(hi)CD4⁺ HLA-DR⁺ (see FIG. 4A). Tonsillar CD123^(hi) cells werealso uniformly CD4⁺ and had an immunophenotype similar to that of bloodCD123^(hi) cells. This phenotype is also compatible with that shown fortonsillar DCs in previous reports. An important difference between bloodand tonsillar cells was, however, that tonsillar CD123^(hi) cells hadalmost undetectable levels of L-selectin. The cells shared the growthpattern of CD123⁺ CD4⁺ cells from bone marrow and blood.

On sections, CD123 bright cells were found scattered throughout theextrafollicular areas, suggesting that these cells might be DCs of theinterdigitating type. A similar staining pattern was observed for CD36,which specifically identified DCs in preparations of mononuclear cells.CD123 also stained endothelium, but these cells are CD4⁻. TheCD123^(hi)CD4⁺ cells were also found at low frequencies (0.1-0.2%) inadult spleen.

Dendritic cells may play an important role in the development of lymphnodes as mice deficient in Rel B lack DCs and are deficient in lymphnodes. An analysis of human fetal lymph nodes for the presence ofCD123^(hi)CD4⁺ cells was therefore conducted. In agreement with thehypothesized function in lymph node development, we found a highpercentage (3-4%) of CD123^(hi) CD4⁺ HLA-DR⁺ cells in fetal lymph nodes(FIG. 4B). The frequency of thymic CD123^(hi)CD4⁺ cells was, however,low, constituting 0.2-0.3% of the mononuclear cells.

Thus, a population of interleukin-3 receptor^(hi) (CD123^(hi))CD4⁺ CD34⁺bone marrow progenitor cells of myeloid origin differentiate intodendritic cells. Interleukin-3R^(hi)CD4⁺ cells with highly similarcharacteristics to the bone marrow population, are furthermore readilydetected among freshly isolated mononuclear cells from peripheral blood,tonsil, thymus, adult and fetal lymph nodes. The frequencies wereparticularly high in fetal lymph nodes, suggesting an important role inthe ontogeny of lymphoid tissues. The possibility to identify and linkthe identity of dendritic cells in blood, bone marrow and lymphoidtissues should facilitate the understanding of the role of dendriticcells in normal and pathological regulation of the immune system.

It is apparent that many modifications and variations of this inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only, and the invention is limited only by the terms of theappended claims.

What is claimed is:
 1. A method of enriching for CD11c⁻ dendritic cellsfrom a population of peripheral blood mononuclear cells, comprising:contacting said population with an anti-CD123 antibody; and thenselecting the cells that are CD123⁺.
 2. The method of claim 1, whereinsaid anti-CD123 antibody is detectably labeled.
 3. The method of claim2, wherein said label is fluorescent.
 4. The method of claim 3, whereinsaid selection is flow cytometric selection.
 5. The method of claim 3,wherein said label is selected from the group consisting of fluoresceinisothyocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE) andperidinin chlorophyll protein (PerCP).
 6. A method of distinguishingCD11c⁻ dendritic cells in a population of peripheral blood mononuclearcells comprising: contacting said population with an anti-CD123antibody, an anti-HLA-DR antibody, an anti-CD4 antibody and at least oneantibody with specificity for a non-dendritic cell lineage marker; anddetecting the cells that are CD123⁺HLA-DR⁺CD4⁺ and negative forexpression of said non-dendritic cell lineage marker.
 7. The method ofclaim 6, wherein each of said non-dendritic cell lineage marker isselected from the group consisting of: CD3, CD14, CD16, CD19, CD56, andCD64.
 8. The method of claim 6, wherein said anti-CD123 antibody isdetectably labeled.
 9. The method of claim 8, wherein said label isfluorescent.
 10. The method of claim 9, wherein said selection is flowcytometric selection.
 11. The method of claim 9, wherein said label isselected from the group consisting of fluorescein isothyocyanate (FITC),allophycocyanin (APC), phycoerythrin (PE) and peridinin chlorophyllprotein (PerCP).
 12. A method of distinguishing CD11c⁻ dendritic cellswithin a population of peripheral blood dendritic cells, comprising:contacting said population with an anti-CD123 antibody; and thendetecting the cells that are CD123⁺.
 13. The method of claim 12, whereinsaid anti-CD123 antibody is detectably labeled.
 14. The method of claim13, wherein said label is fluorescent.
 15. The method of claim 14,wherein said selection is flow cytometric selection.
 16. The method ofclaim 14, wherein said label is selected from the group consisting ofFITC, APC, PerCP, and PE.
 17. A method of depleting CD11c⁻ dendriticcells from a population of peripheral blood mononuclear cells,comprising: contacting said population with an anti-CD123 antibody; andthen removing the cells that are CD123⁺.
 18. The method of claim 17,wherein said anti-CD123 antibody is detectably labeled.
 19. The methodof claim 18, wherein said label is fluorescent.
 20. The method of claim19, wherein said selection is flow cytometric selection.
 21. The methodof claim 19, wherein said label is selected from the group consisting ofFITC, APC, PerCP, and PE.
 22. In a mixed lymphocyte reaction, theimprovement comprising: incubating the lymphocytes in said reaction withdendritic cells enriched for CD123⁺CD11c⁻ dendritic cells.